The present invention relates to biosensors and methods involving the use of these biosensors in detecting the presence of enzymes by detecting their enzymatic activity.
A number of proteins which are useful as immunodiagnostic analytes and disease markers have the additional property of enzymatic activity, in particular protease activity. In addition, other classes of proteins exhihibit nuclease activity.
Prostate Specific Antigen (PSA), a diagnostic marker for prostate cancer is an example of a protein which exhibits protease activity, and belongs to the class of proteins known as the serine proteases. Examples of other proteases which are important immunodiagnostic markers include blood coagulation enzymes, elastase, cathepsin B.
There are also a number of important industrial enzymes such as subtilisin, papain and xcex1-amylase.
Examples of important nucleases are restriction enzymes, e.g., BamH1. Hind III, polymerases which can act as nucleases under certain conditions. e.g., T4 DNA polymerase, reverse transcriptase, which acts as an Rnase under certain conditions, e.g. Rnase H, and exo- and endo-nucleases, e.g. S1 nuclease.
Current diagnostic tests employ immunoassays for the detection of PSA (e.g. a number of analytical instruments such as Abbott""s AXsym. Boehringer Mannheim""s Elecsys, and CIBA-Corning""s ACS-180. all have ELISA-based PSA tests). These tests use antibodies raised against the PSA molecule which recognise the specific epitope sites within the protein molecule.
A variation on these approaches is disclosed in International Patent application No. PCT/AU95/00536. In this reference there is disclosed a range of substrates specifically cleaved by PSA. There is also disclosure in this reference of an assay system for proteases such as PSA which make use of the activity of the protease. This assay system involves the use of a ligand to capture the PSA and the subsequent use of a substrate for the PSA.
The present inventors have developed devices and methods for the detection of enzymes which make use of the protein""s protease activity. These devices and methods involve the use of membrane based biosensors. Information regarding such biosensors can be found in International Patent Application Nos PCT/AU88/00273, PCT/AU89/00352, PCT/AU90/00025, PCT/AU92/00132, PCT/AU93/00509, PCT/AU93/00620, PCT/AU94/00202 and PCT/AU95/00763. The disclosure of each of these applications is included herein by reference.
The present invention involves providing a substrate for the enzyme to be detected and then sensing the digestion of the substrate by the enzyme. This may be achieved in a number of ways, for example the digestion of the substrate may remove a group from the ionophore thereby releasing the ionophore so that it diffuses laterally within the membrane or may result in an increase in the ability of ions to pass through the ionophore simply by a reduction in xe2x80x9cstericxe2x80x9d hindrance. Alternatively the digestion of the substrate when attached to a membrane spanning component may result in the release of the ionophore such that it may diffuse laterally within the membrane Clearly this could also be achieved by digestion of substrates attached to both the ionophore and membrane spanning component.
In another arrangement the digestion of the substrate results in the release of ionophore including probe which then inserts itself into the membrane.
Accordingly, in a first aspect the present invention consists in a biosensor for use in detecting the presence of an enzyme in a sample, the biosensor comprising a membrane and means for determining the impedance of the membrane, the membrane having ionophores therein to which are attached linkers, the linkers being cleavable by the enzyme to be detected, the cleavage of the linker causing a change in the ability of ions to pass through the membrane via the ionophores.
In a preferred embodiment of the present invention the linker is attached to the membrane such that the ionophore is prevented from diffusing laterally within the membrane. It is preferred that the linker is attached to membrane spanning components provided in the membrane. This attachment may be achieved in a number of ways such as covalent attachment, however, it is presently preferred that the attachment is achieved by providing on each of the linker and membrane spanning component one member of a ligand binding pair. A preferred ligand binding pair is biotin streptavidin. In another preferred arrangement both the membrane spanning component and the linker are provided with moieties which are both bound to the same molecule, for example biotin is provided on both the membrane spanning component and the linker and there is cross-linking via streptavidin.
The moiety on the membrane spanning component may also be attached via a linker. This may be the same linker as that provided on the ionophore or may be different.
In a further preferred embodiment the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules, a plurality of ionophores and a plurality of membrane-spanning lipids prevented from lateral diffusion in the membrane, the ionophores comprising first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer, the first half membrane spanning monomers being prevented from lateral diffusion in the first layer, the second half membrane spanning monomers being linked to the membrane spanning lipids via the linker. Following cleavage of the linker by the enzyme the second half membrane spanning monomers can diffuse laterally within the second layer independent of the first half membrane spanning monomers.
In a second aspect the present invention consists in a biosensor for use in detecting the presence of an enzyme in a sample, the biosensor comprising a membrane and means for determining the impedance of the membrane, the membrane having a plurality of ionophores and a plurality of membrane-spanning components therein, the membrane-spanning components having attached thereto linker molecules to which are connected the ionophores, the linker molecules being cleavable by the enzyme to be detected, the cleavage of the linker molecules causing a change in the ability of ions to pass through the membrane via the ionophores.
In a preferred embodiment the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules and the membrane-spanning components are prevented from lateral diffusion in the membrane. Preferably the ioniophores comprise first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer with the first half membrane spanning monomers being prevented from lateral diffusion in the first layer. The second half membrane spanning monomers are connected to the membrane-spanning components via the linker molecule.
The ionophores in both these aspects are preferably gramicidin or analogues thereof.
While a range of enzymes can be detected using the biosensor or the present invention the biosensor is particularly useful in the detection of proteases, in particular those of clinical importance such as PSA, fibrinogen etc.
In a third aspect the present invention consists in a biosensor for the detection of enzymes comprising first and second zones, means to allow addition of a sample suspected to contain an enzyme to the first zone, the first zone containing a probe linked to a carrier via a linker cleavable by the enzyme and means to allow passage of unlinked probe from the first zone to the second zone; the second zone including a membrane the impedance of which is dependent on the presence or absence of the probe and means to measure the impedance of the membrane.
In a preferred embodiment of this aspect of the present invention the membrane comprises a first and a second layer of a closely packed array of amphiphilic molecules and a plurality of ionophores comprising a first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer. The second half membrane spanning monomers being capable of lateral diffusion within the second layer independent of the first half membrane spanning monomers, the first half membrane spanning monomers being prevented from lateral diffusion in the first layer, and a ligand provided on at least the second half membrane spanning monomers, said ligand being reactive with the probe or a portion thereof, the binding of the probe to the ligand causing a change in the relationship between the first half membrane spanning monomers and the second half membrane spanning monomers such that the flow of ions across the membrane via the ionophores is allowed or prevented.
In a preferred embodiment the probe includes streptavidin and the ligand includes biotin.
In yet another preferred embodiment the probe includes an ionophore such that when the probe comes into contact with the membrane the ionophore inserts itself into the membrane changing the impedance of the membrane, As an example of such an arrangement the probe may include valinomycin which inserts itself into the membrane.
In a preferred embodiment of the present invention the enzyme to be detected is a protease in particular Prostate Specific Antigen. In this case it is preferred that the linker or linker molecule includes the sequence Ala-Val-Tyr.
As will be recognised by those skilled in the art the actual linker used will depend on the enzyme to be detected. Examples of some enzymes and their corresponding substrates are set out in Whittaker et al. Analytical Biochemistry: 220, 238-243 (1994), the disclosure of which is incorporated by cross-reference.
In a further aspect the present invention consists in a method of detecting the presence of an enzyme in a sample comprising adding the sample to the biosensor of the first or second or third aspect of the present invention and measuring the change in impedance of the membrane.
As will be readily apparent the biosensors and methods of the present invention do not detect total enzyme: they detect only active enzyme. This is important as in a number of situations it is the amount of active enzyme present which is of importance not simply the total amount of enzyme present as would be measured in a standard sandwich ELISA.
It will also be apparent that the sensors of the present invention can be used to detect a wide range of enzymes. These enzymes include nucleases, protease amylases etc. The sensors are adapted to the particular enzyme to be detected by adjusting the make-up of the linker. For example to detect proteases the linker will typically include a peptide portion which is cleaved by the enzyme. Information regarding peptide sequences cleaved by specific proteases is provided in Whittaker et al referred to above, Where the enzyme to be detected is a nuclease the linker will typically include a nucleic acid sequence. Information regarding specific sequences cleaved by specific enzymes can be found in xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d Ausebel et al (1987) John Wiley and Sons, N.Y.
The sensors of the present invention may also find use in drug development for determining DNA-drug binding sites. The sensors could also be used in determining DNA-protein binding sites. The sensors may also find use in diagnosing infection. For example the sensors could be used to detect enzyme activity specifically associated with a pathogen.
Industrially and clinically relevant proteases and substrates include thrombin and serine proteases including PSA. A list of lysis enzymes is found in xe2x80x9cSpecificity of Proteolysisxe2x80x9d Borivoj Keil (1992) Springer Verlag N.Y. pp. 283-323. Useful ones are the serine and cysteine proteases. See also xe2x80x9cProteolytic Enzymesxe2x80x9d: a Practical Approachxe2x80x9d R. J. Benyon and J. S. Bond (eds) 1989 Oxford University Press N.Y. p232, pp. 241-249. Commercially significant proteases and protease inhibitors for which the present technology is relevant are available in serine, cysteine, aspartic and metallo types. The serine proteases include the endoproteinase-Arg-C, -Glu-C, Lys-C, factor Xa, proteinase K. subtilisin and trypsin, and the exopeptidases acylamino-acid-releasing enzyme, carboxypeptidase P, and carboxypeptidase Y. The cysteine proteases include the endopeptidases bromelain, cathepsin B. clostripain, papain, and the exopeptidases cathepsin C and pyroglutamate aminopeptidase. The aspartic proteases include the endopeptidases cathepsin D and pepsin. The metallo proteases include the endopeptidase thermolysin and the exopeptidases aminopeptidase M, carboxypeptidase-A, -B and leucine aminopeptidase. The listing is not intended to be exclusive and indicates the broad utility of the present invention. Other commercially useful proteases are listed in the publications cited above, which are included herein by reference. For example it also includes the endopeptide endoproteinase-Asp-N of unknown type.